Plant lectins as mucosal adjuvants

ABSTRACT

The invention provides a method of increasing an immune response in a mammal. The method involves administering to the mammal an admixture comprising an immunogen and a plant lectin. The plant lectin acts as an adjuvant to increase an immune response against the immunogen. The method is especially well-suited for mucosal administration to humans and other mammals.

This application claims the benefit of provisional application Ser. No.60/161,371 filed Oct. 26, 1999, which is incorporated herein byreference.

TECHNICAL FIELD OF THE INVENTION

The invention relates to the enhancement of an immune response in amammal. More particularly, the invention relates to the use of plantlectins as adjuvants.

BACKGROUND OF THE INVENTION

Because most pathogens colonize and invade the host at mucosal surfaces,the induction of immunity at these sites is a rational and attractiveapproach to prevent infection (1). Mucosal routes for vaccine deliveryare non-invasive, so administration is relatively simple andinexpensive. Furthermore, the potential to induce a range of mucosal andsystemic immune responses after mucosal vaccine delivery allows thepossibility of effective immunization against many diseases. Forexample, specific IgA alone can protect mice against intranasalinfection with influenza (2) and intestinal infection with Vibriocholerae (3). However, mucosal delivery of nonreplicating immunogenstypically does not stimulate strong immune responses. Where responsesare induced, the delivery of multiple high doses is often necessary (4).In addition, mucosal delivery of immunogens frequently results insystemic unresponsiveness (1).

A number of strategies may be used to enhance responses to mucosallydelivered vaccines. Live bacterial and viral vectors which colonize themucosae can be used to deliver immunogens (5). Imparting particulatecharacteristics to immunogens by association with biodegradablemicroparticles (6) or liposomes (7) can also enhance mucosalimmunogenicity.

Another approach is the use of lectin-like molecules with adjuvantproperties. The most powerful mucosal adjuvants identified to date arecholera toxin produced by Vibrio cholerae (CT) and heat-labileenterotoxin (LT) from enterotoxigenic strains of Escherichia coli (8,9). CT and LT are well-characterized mucosal immunogens and adjuvantsfor bystander proteins. These toxins contain separate A and B subunits(referred to as CTA and CTB, respectively). The B subunits mediatebinding to cell surface receptors (20). GM1 ganglioside is considered tothe principal receptor for CT (21), but CTB may bind to cell surfacereceptors other than GM1 (22). After binding of the B subunit, the Asubunit reaches the cytosol and activates adenyl cyclase leading to alarge increase in [cAMP]_(i) (10, 11). LT is structurally andfunctionally similar to CT and is comparable to CT as a systemic ormucosal adjuvant (23, 24). In mice, CT strongly stimulates humoral andcell-mediated immune responses, including mucosal IgA production andcytotoxic T cell effector functions (10). Stimulation of toxin-specificlocal and systemic responses and responses to co-administered immunogensdistinguish these molecules from most soluble proteins which are poorlyimmunogenic when administered mucosally (10, 11). The toxicity of thesemolecules, however, prevents clinical application.

Certain plant lectins have been investigated as agents for specifictargeting of molecules to a mucosal epithelium. Plant lectins areproteins containing at least one non-catalytic domain, which bindsspecifically and reversibly to a monosaccharide or oligosaccharide (13).For example, Giannasca et al. (14) discloses that intranasalimmunization with a lectin-immunogen conjugate stimulated induction ofspecific IgG antibodies, while immunogen alone or admixed with lectindid not. U.S. Pat. No. 4,470,967 discloses that a complex of aglycoprotein immunogen with a lectin can act as an adjuvant to increasethe immune response against the immunogen. Similarly, WO 86/06635discloses a chemically modified immunogen-lectin complex which can beused to elicit an immune response in vertebrates, including mammals. Ineach of these cases, however, the lectin was physically coupled to theimmunogen. This requires at least one extra preparation step and mayactually alter an epitope of the immunogen against which an immuneresponse is desired, such as an epitope against which a neutralizingimmune could be directed.

Thus, there is a need in the art for simple, effective, and non-toxicmethods of increasing immune responses in a mammal, particularly aftermucosal administration, without the need to complex the immunogen withanother molecule and potentially mask or alter desirable epitopes.

SUMMARY OF THE INVENTION

The invention provides a method of increasing an immune response in amammal by administering to the mammal an admixture comprising animmunogen and a plant lectin. The mammal thereby produces an immuneresponse which is increased relative to an immune response produced inthe absence of the plant lectin.

The invention thus provides a simple and effective method of increasingan immune response in a mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Bar graph showing total IgA levels (ng/ml) measured innasotracheal washes of mice after four intranasal doses of immunogen.

FIGS. 2A-D. Plots showing the adjuvant effect of plant lectins.OVA-specific serum IgG antibody titers from mice immunized intranasally.FIG. 2A, serum IgG titers after one dose (day 13). FIG. 2B, serum IgGtiters after two doses (day 27). FIG. 2C, serum IgG titers after threedoses (day 41). FIG. 2D, serum IgG titers after the final dose (day 56).

FIG. 3. Plot showing OVA-specific serum IgG1 antibody titers measured inmice immunized intranasally.

FIGS. 4A-D. Plots showing OVA-specific IgA antibody titers measured insecretions of mice immunized intranasally. FIG. 4A, saliva; FIG. 4B,vaginal wash; FIG. 4C, nasotracheal wash; FIG. 4D, intestinal wash.

FIGS. 5A-F. CT/plant lectin-specific serum IgG antibody titers measuredin mice immunized intranasally. FIG. 5A, CT; FIG. 5B, LEA; FIG. SD, PHA;FIG. 5E, WGA; FIG. 5F, UEA-I.

FIGS. 6A-D. Plots showing gD2-specific serum IgG antibody titers frommice immunized intranasally. FIG. 6A, day 14; FIG. 6B, day 28; FIG. 6C,day 41; FIG. 6D, day 55.

FIGS. 7A-D. Plots showing gD2-specific serum IgG subclass antibodytiters. FIG. 7A, IgG1; FIG. 7B, IgG2a; FIG. 7C, IgG2b; FIG. 7D, IgG3.

FIGS. 8A-D. Plots showing gD2-specific IgA antibody titers measured insecretions. FIG. 8A, saliva; FIG. 8B, vaginal wash; FIG. 8C,nasotracheal wash; FIG. 8D, intestinal wash.

FIG. 9. gD2-specific total serum IgG and IgG subclass titers from miceimmunized intransally on days 1, 21, and 42 with either gD5 (5:g) aloneor gD2 (5:g) together with 1:g of CT, ML-I, Nigrin B, Basic Nigrin B,Ebulin r1, SNA II or SELfd. FIG. 9A, IgG; FIG. 9B, IgG1; FIG. 9C, IgG2a;FIG. 9D, IgG2b.

FIG. 10. gD2-specific IgA antibody titers measured in secretions of miceimmunized intranasally on days 1, 21, and 42 with either gD2 (5:g) aloneor gD2 (5:g) together with 1:g of CT, ML-I, Nigrin B, Basic Nigrin B,Ebulin r1, SNA II or SELfd. FIG. 10A, saliva; FIG. 10B, vaginal wash;FIG. 10C, nasotracheal wash; FIG. 10D, gut wash.

FIG. 11. gD2-specific serum IgA and IgG antibody titers measured in miceimmunized intranasally on days 1, 21, and 42 with either gD2 (5 μg)alone or gD2 (5 μg) together with 1 μg of CT, ML-I, or UEA-1. FIG. 11A,serum IgA; FIG. 11B, serum IgG.

FIG. 12. gD2-specific IgG subclass antibody titers measured in miceimmunized intranasally on days 1, 21, and 42 with either gD2 (5 μg)alone or gD2 (5 μg) together with 1 μg of CT, ML-I, orUEA-1. FIG. 12A,IgG1; FIG. 12B, IgG2a; FIG. 12C, IgG2b.

FIG. 13. gD2-specific IgA antibody titers measured in secretions of miceimmunized intranasally on days 1, 21 and 42 with either gD2 (5 μg) aloneor gD2 (5 μg) together with 1 μg of CT, ML-I, or UEA-1. FIG. 13A,saliva; FIG. 13B, vaginal wash; FIG. 13C, gut wash; FIG. 13D,nasotracheal wash.

FIG. 14. Mean concentrations of IL-5, WL4, and IFN production and countsper minute for T cell proliferation assay. FIG. 14A, IL-5 production inspleen cells; FIG. 14C, IL-4 production in spleen cells; FIG. 14E, IFNproduction in spleen cells; FIG. 14B, IL-5 production in cervical lymphnodes; FIG. 14D, IL4 production in cervical lymph nodes; FIG. 14F, IFNproduction in cervical lymph nodes; FIG. 14G, T cell proliferation byspleen cells. Responses were measured at week 8 after threeimmunizations (days 0, 21, 42) with gD2, MLI, or UEA-1, or with gD2+MLI,UEA-1, or LTK63. Spleen cells and cervical lymph node cells wereisolated and stimulated in vitro with gD2 (0 μg/ml, 1 μg/ml, or 5 μg/ml)or with gD2 coupled to latex beads diluted 1:1000 or 1:5000 or withPMA/cd3

FIG. 15. OVA-specific serum IgG antibody titers from mice immunized bygavage on days 1, 14, 28 and 49 with either OVA (5 mg) alone or OVA (5mg) together with CT (10 μg), ML-I (10 μg), ML-II (10 μg) or ML-III (10μg). FIG. 15A, serum IgG titers after one dose (day 13; (FIG. 15B, serumIgG titers after two doses (day 27); FIG. 15C, serum IgG titers afterthree doses (day 48; FIG. 15D, serum IgG titers after the final dose(day 62).

FIG. 16. OVA-specific serum IgG subclass and IgA antibody titersmeasured in mice immunized by gavage on days 1, 14, 35, and 49 witheither OVA (5 mg) alone or OVA (5 mg) together with CT (10 μg), ML-I (10μg), ML-II (10 μg), or ML-III (10 μg). FIG. 16A, IgG1; FIG. 16B, IgG2a;FIG. 16C. IgG2b; FIG. 16D, IgG3.

FIG. 17. OVA-specific IgA antibody titers measured in secretions of miceimmunized by gavage on days 1, 14, 35 and 49 with OVA (5 mg) alone orOVA (5 mg) together with CT (10:g), ML-I (10:g), ML-II (10:g), or ML-III(10:g). FIG. 17A, saliva, FIG. 17B, vaginal wash; FIG. 17C, nasotrachealwash, FIG. 17D, intestinal wash.

FIG. 18. Western blot showing WGA in gut homogenates collected from mice6 hours following gavage with a single dose of 1 mg WGA.

FIG. 19. Western blots showing homogenized stomach tissues and smallintestine washings collected 1 hour after gavage with a single dose of 1mg PHA.

FIG. 20. Western blot showing UEA1 in extracted mouse kidney tissuescollected 24 hours following gavage with a single dose of 1 mg of UEA1.

FIG. 21. Western blot showing WGA in mouse liver tissues collected 24hours following gavage with a single dose of 1 mg WGA.

FIG. 22. Western blot showing the susceptibility of native andkidney-extracted UEA1 to digestion by trypsin.

DETAILED DESCRIPTION

It is an aspect of the present invention that certain plant lectins actas mucosal adjuvants to increase immune responses, including anincreased antibody titer, against a variety of immunogens, thuspermitting simple, non-toxic, and cost-effective vaccine or immunogeniccompositions to be prepared. Vaccine or immunogenic compositions of theinvention are admixtures comprising a plant lectin and an immunogen.Such admixtures are especially suitable for mucosal delivery to mammals,including humans, and are thus useful for veterinary as well as humanmedical purposes.

Admixtures of the invention comprise a plant lectin and an immunogen.The immunogen and the lectin are not coupled together chemically, butare simply mixed together in an appropriate liquid medium, such asphosphate buffered saline or other isotonic saline solution. Optionally,an admixture can comprise stabilizing agents, including antimicrobialagents, preservatives, and the like. The proportions of immunogen andlectin in the admixture can be varied, such as at least about 1:1, 2:1,3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1, depending on the particularimmunogen and lectin combination selected. If desired, at least 2, 3, 4,or more different immunogens and/or lectins in varying proportions canbe included in an admixture.

Lectins useful in the invention include plant lectins such as mistletoelectin I (ML-1), mistletoe lectin II (ML-II), mistletoe lectin III(ML-III), wheat germ agglutinin (WGA), and Ulex europaeus (UEA-1). Otherlectins which may be useful include lentil bean lectin, jack bean lectin(concanavalin A), and asparagus pea, broad bean, camel's foot tree,castor bean, fava bean, hairy vetch, horse gram, Japanese wisteria,Jequirity, Scotch labumum, lima beam, lotus, mung bean, Osage orange,Pagoda tree, garden pea, potato, red kidney bean, Siberian pea tree,spindle tree, sweet pea, tomato, and winged pea lectins.

Type 2 ribosome inactivating proteins (RIP), such as nigrin b, basicnigrin b, ebulin 1, ebulin r, ebulin f, nigrin f, SNA1, SNA1′, SNAV,SNAVI, Sambucus nigra SNLRP1, SNLRP2, ricin, Ricinus lectin, PolygonatumRIP, Sieboldin-6, abrin, abrin 11, modeccin, volkensin, SSA, Cinnamonin,porrectin, gelorin, Evanthis hyemalis, RIP, Iris agglutinin, ML-I,ML-II, and ML-III, are especially useful as adjuvants. Such lectinscontain an N-glycosidase A subunit responsible for theribosome-inactivating activity and a galactose-specificcarbohydrate-binding B subunit (29). ML-I, ML-II, and ML-III are strongmucosal adjuvants, which can stimulate high antibody titers in sera andmucosal secretions. Type 2 RIPs which do not show in vivo toxicity, suchas ebulin-1 (32), nigrin b (33) and basic nigrin b (34), areparticularly useful. Alternatively, lectins can be genetically“detoxified,” for example by modifying one or more amino acids bysite-directed mutagenesis such that the lectins retain their adjuvantproperties but are non-toxic to the mammalian recipient (see 35-39; EP0880361; EP 620850; EP 95/903889.4).

Lectins in an admixture are preferably in an unbound, water-solubleform. Suitable lectins for use in admixtures of the invention can bepurchased from commercial suppliers, such as Sigma. Alternatively,lectins can be purified using protein purification protocols well knownin the art, including size exclusion chromatography, ammonium sulfatefractionation, ion exchange chromatography, affinity chromatography,crystallization, electrofocusing, and preparative gel electrophoresis.

Immunogens against which a cellular and/or humoral response can beincreased using a plant lectin adjuvant include proteins of infectiousagents, such as viruses, bacteria, mycoplasmas, prions, and yeast, aswell as hormones, allergens such as grass, weed, tree, and plantpollens, epithelia of animals such as cats, dogs, rats, and pigs, housedust, and wheat chaff. Means of obtaining such immunogens are well knownin the art. An immunogen need not be able to raise a cellular and/orhumoral response in the absence of the plant lectin.

Admixtures of the invention can be administered to a recipient mammal ina variety of formulations. For example, admixtures can be entrapped inor adsorbed to the surface of microparticles, such aspoly(lactide-co-glycolides) (PLG) (35; U.S. Pat. Nos. 5,804,212,6,876,761, and 5,603,960; PCT/US99/17308). Admixtures can also beadministered in conjunction with bioadhesive polymers, such as thosedescribed in PCT/US99/12105, PCT/US99/11906, and U.S. Pat. Nos.5,955,097, 5,800,832, 5,744,155, and 5,814,329. Alternatively, entericformulations of admixtures can be used for oral administration (see U.S.Pat. No. 5,968,554).

An admixture of the invention can be administered to a mammal byinjection, i.e., subcutaneous, intramuscular, or other parenteralinjection, such as transdermal or transcutaneous injection, by oralingestion, or by intranasal administration. Admixtures can beadministered to any mammal in which it is desired to increase an immuneresponse, including but not limited to rats, cats, dogs, rabbits,horses, cows, mice, guinea pigs, chimpanzees, baboons, and humans.

Mucosal administration, particularly intranasal administration intoeither one or both nostrils, is preferred. Doses can be delivered, forexample, in one or more drops or using a spray, such as an aerosol ornon-aerosol spray. If desired, multiple administrations of an admixturecan be used to increase antibody titers against a particular immunogen.Intervals between multiple administrations can be at least 1, 2, 3, 4,5, 6, or 7 or more days, or at least 2, 3, or 4 or more weeks, dependingon the particular immunogen and/or lectin in the admixture. The volumeof admixture to be administered will vary according to the mode ofadministration and size of the mammal. Typical volumes for in nasaladministration vary from at least 5, 10, 15, 25, 50, 75, 100, 200, or250:1, to at least 500:1 or more per intranasal dose.

The concentration of immunogen in an admixture also will vary accordingto the particular immunogen and route of administration selected. Forintranasal administration, for example, the concentration of animmunogen in an admixture varies from at least 0.033, 0.67, 0.1, 0.2,0.33, 0.5, 0.67, 0.75, 1, 2, 2.5, 5, 7.1, 10, 12.5, 15, 17.5, 20, or25:g/:1.

Admixtures of the invention preferably increase antibody production aswell as T cell responses, including cytokine production, target-cellkilling, macrophage activation, B-cell activation, and lymphokineproduction. Admixtures of the invention preferably increase a T cellresponse or an antibody titer by at least 10, 15, 20, 25, 30, 40, 50,75, or 100 percent or more relative to such responses to the immunogenalone in the absence of the plant lectin.

Methods of measuring T cell responses are well known in the art. (SeeJaneway et al., eds., 1997, IMMUNOBIOLOGY: THE IMMUNE SYSTEM IN HEALTHAND DISEASE, 3d ed., at pages 2:31-2-33; Abbas et al., 1997, CELLULARAND MOLECULAR IMMUNOLOGY, 3d ed., at pages 250-277 and 290-293).

According to the invention, antibodies can be produced which aredirected against the immunogen in the admixture. Antibodies whichspecifically bind to the immunogen typically provide a detection signalat least 5-, 10-, or 20-fold higher than a detection signal providedwith other proteins when used in immunochemical assays, such as Westernblots, ELISAs, radioimmunoassays, immunohistochemical assays,immunoprecipitations, or other immunochemical assays known in the art.Preferably, antibodies which specifically bind to a particular immunogendo not detect other proteins in immunochemical assays and canprecipitate the immunogen from solution.

Antibody titer is preferably measured by ELISA, as described in Example1, below. IgG, including IgG subtypes IgG1, IgG2a, IgG2b, and IgG3, aswell as IgA antibodies directed against the immunogen can be measured inserum, in saliva, and in mucosal secretions, including vaginal, nasal,and gut washes (see Example 1).

The complete contents of all patents and patent applications cited inthis disclosure are expressly incorporated herein.

The following examples are provided for exemplification purposes onlyand are not intended to limit the scope of the invention which has beendescribed in broad terms above.

EXAMPLE 1

Materials and Methods.

Immunogens and Lectins.

Cholera toxin (CT), ovalbumin (OVA, type V, hen egg) and WGA wereobtained from Sigma (Poole, UK). PHA from kidney bean was prepared asdescribed previously (15). UEA-I and LEA were obtained from VectorLaboratories. ML-I was isolated as described previously (16).

Animals.

Eight week old female Balb/c mice (Harlan Olac, Bicester, UK) were givenfree access to commercial stock diet (Labsure, Manea, UK) and water.

Mucosal Immunization Schedule.

Groups of mice (n=10) were bled one week prior to the firstimmunization. On days 1, 14, 28, and 42, mice were immunizedintranasally with PBS, OVA (10:g) alone, or OVA (10:g) mixed with CT(1:g), ML-I (1:g), LEA (10:g), PHA (10:g), WGA (10:g), or UEA-I (10:g).In other examples, mice were immunized intranasally with 5:gglycoprotein D2 (gD2) from Herpes simplex virus type 2 on days 1, 14,28, and 49 alone or in an admixture with 1:g of either CT, ML-I, ML-II,or ML-III. Mice were dosed with 30:1 of each preparation (15:1 pernostril) through fine tips attached to a pipette.

Collection of Blood and Mucosal Secretions.

Blood samples were collected one day prior to each immunization bybleeding from the tail vein following a 10 minute incubation at 37° C.Two weeks after the final immunization, animals were terminallyanesthetized (hypnorin plus diazepam) to allow collection of salivaryand vaginal secretions. Mice were then killed by anesthetic overdosefollowed by exsanguination. Blood was immediately collected andcentrifuged, and the serum was stored at −20° C.

Absorbent cellulose wicks (Whatman International, UK) were used forcollection of saliva and vaginal fluid as described previously (17).Wash fluid (ice-cold 0.01 M PBS, 50 mM EDTA, 5 mM PMSF, 5:g/mlAprotinin) was used for elution of antibody from wicks and for nasal andintestinal washes. Saliva was collected by the insertion of a wick tipinto the mouth for 2 minutes (17). Antibody was extracted from wicksinto 400:1 mucosal wash fluid. Vaginal fluid was collected by repeatedflushing and aspiration of 50:1 wash fluid and insertion of a wick for 2minutes. Antibody was extracted from wicks into 400:1 wash fluid.Nasotracheal washes were collected from decapitated animals bybackflushing 0.5 ml of mucosal wash fluid from the trachea. Intestinalwashes were obtained by flushing the small intestine with 10 ml ofice-cold wash fluid. All secretions were stored at −20° C. untilrequired for analysis.

Detection of Specific Antibodies by ELISA.

ELISAs were set up to enable measurement of specific IgG, IgA, and IgGsubclasses to OVA, CT, and plant lectins. Sera (from 1:100) and mucosalsecretions (from 1:2) were titrated in the appropriate dilution buffer.Microtiter plates (Immunolon 4, Dynatech) were coated with 75:1 ofimmunogen per well (1:g/ml for CT/lectins, 50:g/ml when measuringresponses to OVA and 2:g/ml when measuring responses to gD2) incarbonate-bicarbonate buffer, pH 9.6, and incubated at 4° C. overnight.After washing, plates were blocked with 2% gelatin/dilution buffer andincubated at 37° C. for 1 hour. Plates were washed, and samples wereadded, serially diluted, and incubated at 37° C. for 1 hour.

Biotinylated antiserum in dilution buffer was added and incubated at 37°C. for 1 hour. After further washes, ExtrAvidin® peroxidase (Sigma)diluted 1:750 in dilution buffer was added and incubated at 37° C. for30 minutes. Plates were washed, and 50:1/well of developing solution(TMB microwell peroxidase substrate (1-C), Kirkegaard and PerryLaboratories, Gaithersburg, Md.) was added. Plates were incubated in thedark with shaking at 37° C. for 30 minutes. The reaction was stopped byaddition of 1M H₂SO₄, and the absorbance was read at 450 nm.

ELISA dilution buffers were as follows: CT (PBS+0.1% Tween (PBST)), OVA(PBST), WGA (100 mM N-acetylglucosamine/PBST), PHA (0.1% Fetuin/PBST),UEA-I (30 mM L-fucose/PBST), LEA (Chitin hydrolysate (1:200)(Vector)/PBST), ML-I (100 mM D-galactose/PBST). Working dilutions ofanti-IgG (1:8000) and IgA (1:2600) biotinylated capture antisera (Sigma)were determined after preliminary assays with pre-immune and pooledpositive sera. Working dilutions of IgG subclass antisera (Serotect)were as recommended by the manufacturers (IgG1 (1:4000), IgG2a (1:4000),IgG2b (1:2000), IgG3 (1:2000)). Endpoint titers were determined as thedilution of a serum or mucosal sample giving an OD value of 0.1 unitsgreater than the mean of control samples at the same dilution.

Total IgA was quantified as specific IgA with the followingmodifications: plates were coated with goat anti-mouse IgA (1:8000;∀-chain specific, Sigma), PBST was used as diluent, and 2% gelatin inPBST was used as blocking solution. Total IgA levels were calculatedfrom the linear region of the IgA (IgA kappa, Sigma) standard curve.Total IgA endpoint titers were determined as the dilution of a samplegiving an OD value of 0.1 units greater than buffer alone.

Statistics.

Data are expressed as the mean± standard deviation. An unpairedtwo-tailed t-test was used to test for significance between groups.Where the standard deviations were significantly different betweengroups, a nonparametric test (Kruskal-Wallis test with Dunn's multiplecomparison post test) was used to assess significance. Kruskal-Wallisnonparametric test with Dunn's multiple comparison post test was alsoused to assess significance of the total IgA data.

EXAMPLE 2 The Effect of Immunization on Total IgA Levels in Sera andSecretions

Mice were immunized by the intranasal route on days 1, 14, 28, and 42with either PBS, OVA (10:g) alone, or OVA (10:g) together with CT (1:g),ML-I (1:g), LEA (10:g), PHA (10:g), WGA (10:g) or UEA-I (10:g). Sampleswere collected two weeks after the final immunization. The results areshown FIG. 1. Data represent the mean±SD.

After four intranasal immunizations with CT+OVA there was a significantincrease in the concentration of total nasotracheal wash IgA (p<0.01)compared with all other groups. Co-administration of CT with OVA did notresult in a significant rise in total IgA concentration in sera or theother mucosal secretions sampled. There was no significant effect ofimmunization with any of the plant lectins on total IgA levels in any ofthe secretions or in serum.

EXAMPLE 3 The Adjuvant Effect of Plant Lectins on OVA-Specific SerumAntibody Responses

Mice were immunized intranasally on days 1, 14, 28, and 42 with eitherOVA (10:g), alone or OVA (10:g) together with CT (1:g), ML-I (1:g), LEA(10:g), PHA (10:g), WGA (10:g) or UEA-I (10:g). Sera were collected 1day before each immunization and at the termination of the study. FIGS.2A-D show the results of this experiment. Points refer to individualdata, and the symbol (−) represents the mean titer.

Two weeks after a single immunization, OVA-specific serum IgG wasdetected in 5/10 mice immunized with CT+OVA and 1/10 mice immunized withML-I +OVA but OVA-specific IgG was not detected in the other groups.After a second dose, higher responses were measured with detectableantibody in all mice immunized with CT+OVA (mean titer 40321) and in9/10 mice immunized with ML-I+OVA (mean titer 11090). Of the othergroups, specific IgG was only detected in mice immunized with UEA-I+OVA(mean titer 91).

After four doses, the highest mean IgG titers were in mice immunizedwith CT+OVA, being approximately 286-fold higher than in mice whichreceived OVA alone. The mean titer in the group immunized with ML-I+OVAwas approximately 118-fold higher than in mice which received OVA alone.Titers in mice immunized with PHA+OVA were similar to those in miceadministered with OVA alone. Administration of LEA+OVA resulted in asmall increase in mean titer compared with OVA alone (5-fold). Deliveryof WGA and UEA-I with OVA respectively led to 41- and 51-fold increasesin mean serum IgG anti-OVA titers compared with OVA alone.

In contrast to the groups which received CT+OVA and ML-I+OVA, responsesin the groups immunized with WGA or UEA-I+OVA were highly variable. As aresult, after the final dose only the CT+OVA and ML-I+OVA groups(difference not significant between groups) had mean OVA-specific IgGtiters significantly higher (p<0.001) than the OVA only group. Titers inthese groups were also significantly higher than in the PHA+OVA group(p<0.001).

In contrast to the high levels of specific IgG, very low titers ofOVA-specific serum IgA were detected. In fact, after the final dose,significant levels of OVA-specific serum IgA were only detected in miceimmunized with CT+OVA (mean titer, 220) and ML-I+OVA (mean titer, 80).

EXAMPLE 4 OVA-Specific IgG Subclass Patterns

Mice were immunized intranasally on days 1, 14, 28, and 42 with OVA(10:g) alone or OVA (10:g) together with CT (1:g), LEA, (1:g), PHA(10:g), WGA (10:g), or UEA-I (10:g). Samples were collected two weeksafter the final immunization. FIG. 3 shows the results of thisexperiment. Points refer to individual data, and the symbol (−)represents the mean titer.

Analysis of the subclass profile of OVA-specific IgG antibodiesindicated a very biased response. The IgG1 titers were similar to thetiters of OVA-specific IgG in most groups. In mice immunized with CT+OVAand ML-I+OVA respectively, the mean titers were approximately 450-foldand 255-fold higher than in mice immunized with OVA alone. Titers in theCT+OVA group were significantly higher than in all groups exceptML-I+OVA (p<0.05). Titers in the ML-I+OVA group were significantlyhigher than in groups which received OVA alone or PHA+OVA (p<001).

OVA-specific IgG2a was detected in 8/10 and 2/10 mice immunized withCT+OVA (mean titer 561) and ML-I+OVA (mean titer 331), respectively.Specific IgG2a was not detected in the other groups. Specific IgG2b wasonly detected in 2/10 mice immunized with CT+OVA and in none of theother groups. Specific IgG3 was not detected. These data are strikinglydifferent to the CT-specific IgG isotype responses in these mice whererelatively high titers of specific IgG2a and significant levels of IgG2band IgG3 were detected (Table 1).

EXAMPLE 5 The Adjuvant Effect of Plant Lectins on OVA-Specific MucosalIgA Responses

Mice were immunized intranasally on days 1, 14, 28, and 42 with OVA(10:g) alone or OVA (10:g) together with CT (1:g) ML-I (1:g), LEA(10:g), PHA (10:g), WGA (10:g), or UEA-I (10:g). Titers were measuredtwo weeks after the final immunization. The results are shown in FIGS.4A-D. Points refer to individual data, and the symbol (−) represents themean titer.

Specific IgA was detected at all mucosal sites sampled in mice immunizedwith CT+OVA and ML-I+OVA. There was no significant difference betweenthe two groups. OVA-specific salivary IgA was not detected in miceimmunized with OVA alone, LEA+OVA or PHA+OVA, but was detected in 2/10and 4/10 mice immunized with WGA+OVA and UEA-I+OVA, respectively. Incontrast, specific salivary IgA was measured in 9/10 and 10/10 miceimmunized with CT+OVA and ML-I+OVA, respectively, with a two-fold highermean titer in the CT+OVA group.

In vaginal washes, OVA-specific IgA was detected in 9/10 and 7/10 miceimmunized with CT+OVA and ML-I+OVA, respectively. The mean titer wasfour-fold higher in the CT+OVA group, but this was largely the result ofone high responder. OVA-specific vaginal wash IgA was not detected inmice immunized with PHA+OVA and was detected in 1/10 mice immunized witheither OVA alone, LEA+OVA, or WGA+OVA, and in 3/10 mice immunized withUEA-I+OVA.

High titers of OVA-specific IgA were detected in nasotracheal washesfrom all mice immunized with CT+OVA or ML-I+OVA, with approximately afive-fold higher titer in the CT+OVA group. OVA-specific nasotrachealwash IgA titers were significantly higher (p<0.05) in mice immunizedwith CT+OVA than in all groups except ML-I+OVA.

Remarkably, the OVA-specific nasotracheal wash IgA titers in thesegroups were comparable to the serum IgA titers. Total IgA titers in serafrom mice immunized with CT+OVA and ML-I+OVA were 33-fold and 73-foldhigher than in nasotracheal washes, respectively. Specific IgA wasdetected in the nasotracheal washes of 1/10 mice immunized with OVAalone but not in any mice immunized with PHA+OVA. OVA-specificnasotracheal wash IgA was measured in 7/10 mice immunized with WGA+OVAand 5/10 mice immunized with UEA-I+OVA or LEA+OVA, respectively.

OVA-specific IgA was detected in gut washes from all mice immunized withCT+OVA or ML-I+OVA, with approximately a four-fold higher titer in theCT+OVA group. Titers in these groups were not significantly differentfrom each other but were significantly higher (p<0.05) than in all othergroups. Among the other groups, OVA-specific gut wash IgA was onlydetected sporadically at a maximum titer of 1:2.

EXAMPLE 6 CT/Plant Lectin-Specific Responses

Mice were immunized intranasally on days 1, 14, 28, and 42 with OVA(10:g) together with CT (1:g), ML-I (1:g), LEA (10:g), PHA (10:g), WGA(10:g) or UEA-I (10:g). The results are shown in FIGS. 5A-F. Data arepresented as the mean±SD. Data are titers of specific serum IgG measuredtwo weeks after the final dose of immunogen.

CT-specific serum IgG was detected in all animals after a single dose ofCT+OVA, and titers increased with each subsequent dose. Specificantibodies of all four IgG subclasses were detected in sera after fourdoses (Table 1). The highest titers were of IgG1, although CT-specificIgG2a, IgG2b and IgG3 were also detected. After the final dose,CT-specific serum IgA was detected in all mice, with a mean titer of4481.

Specific IgA was also detected in all animals in saliva, vaginal wash,nasotracheal wash, and gut wash. Salivary IgA titers were relativelyconsistent between animals (approximately 10-fold lower mean titer thanin serum). Total IgA titers in saliva from these mice were 1340-foldlower than in serum. Vaginal IgA titers were highly variable, with asingle high responder increasing the mean titer. High titers ofCT-specific IgA were measured in nasotracheal washes from all animalswith a mean titer comparable to the serum IgA titer. Specific IgA wasalso detected in intestinal washes of all mice, but at a lower meantiter than at the other mucosal sites sampled.

Intranasal delivery of a single dose of ML-I+OVA stimulated theproduction of ML-I specific IgG in 3/10 mice. After the second andsubsequent doses, high titers of specific IgG were detected in all mice(FIG. 5). Analysis of ML-I-specific serum IgG subclasses found hightiters of ML-I-specific IgG1 (Table 1). ML-I-specific IgG2a and IgG2bwere also detected, but specific IgG3 was not detected. ML-I-specificIgA was detected in all mice in serum and at all mucosal sites sampledafter four doses. Titers in the saliva were consistent for all animals,while a single very high responder increased the mean titer in thevaginal washes. High ML-I-specific IgA titers were measured innasotracheal washes of all animals. As with CT, the mean ML-I-specifictiter in nasotracheal washes was comparable with the serum IgA titer(approximately two-fold lower), which was remarkable as the total IgAtiters in nasotracheal washes were 73-fold lower than in sera from thesemice. Specific IgA was also detected in gut washes from all animals.

In mice immunized with LEA+OVA, LEA-specific serum IgG was detected in9/10 mice after a single dose. The titer increased after each subsequentdoes to a relatively high level after the final immunization (FIG. 5).Analysis of IgG subclasses found high titers of LEA-specific IgG1 and alow mean IgG2a titer (Table 1). Specific serum IgA was detected in 7/10mice after four doses, but at a low level. Specific IgA was alsodetected in all four mucosal secretions tested, although in comparisonto the data in the CT+OVA and ML-I+OVA groups the titers were highlyvariable.

PHA-specific serum IgG was detected in 1/10 mice after a single dose ofPHA+OVA. After subsequent doses the titer increased, and specific IgGwas present in 8/10 animals after the final dose (FIG. 5). Of the IgGsubclasses, only specific IgG1 was detected (Table 1). Low titers ofspecific serum IgA were detected in all animals. PHA-specific IgA wasnot detected in saliva or vaginal washes but was detected innasotracheal washes of 5/10 mice and gut washes of 1/10 mice.

The lowest titers of specific antibody were elicited to WGA, even afterfour doses of WGA+OVA (FIG. 5). Specific IgG1 was detected in 2/10 mice,and the other IgG subclasses were not detected (Table 1). Specific IgAwas detected in a number of mice after four doses, but at a maximumtiter of 1:100. Low titers of specific IgA were measured in a smallnumber of mice in saliva, vaginal washes, and nasotracheal washes. Thesedata are in contrast to the OVA-specific data from this group, whererelatively high levels of OVA-specific serum IgG were detected in anumber of mice.

UEA-I-specific serum IgG was not detected after a single dose ofUEA-1+OVA, but was detected after subsequent doses and in 8/10 miceafter the final dose (FIG. 5). Specific IgG1 was detected in 9/10 miceafter the final dose (Table 1), specific IgG2a in 1/10 mice, and IgG2band IgG3 were not detected. Specific serum IgA was detected in 3/10 miceafter the final dose. Relatively low levels of IgA were detected insaliva, vaginal washes, and nasotracheal washes.

The present data indicates that the type of response elicited to theadjuvant and to the immunogen may differ. High titers of specific IgGGwere detected to both OVA and CT, but while relatively high titers ofCT-specific IgG2a were measured, there was little or no OVA-specificIgG2a. Delivery of ML-I+OVA led to similar results, although theML-I-specific IgG2a titers were relatively low. Previous work foundhigher OVA-specific IgG1 than IgG2a titers after delivery of OVA+CT,while higher titers of CT-specific IgG2a than IgG1 were found in thesame mice (24). Feeding mice with CT+keyhole limpet hemocyanin (KLH)stimulated a strong KLH-specific secretory IgA response in mice whichwere high responders to CT with a much smaller effect in poor responders(29). Thus, the oral adjuvant effect of CT depended on a strong immuneresponse to CT itself. However, WGA and UEA-I increased the serum IgGresponse to OVA (through not significantly) and were not highlyimmunogenic. A recent study found that several dietary lectins,including PHA, could trigger human basophils to release IL-4 and IL-13.ConA and PHA-E, for example, induced IL-4 levels as high as thoseobtained by stimulation with anti-IgE antibodies. Lectins thatstimulated high levels of IL-4 also triggered release of IL-13 andhistamine, possibly by inducing IL-4, which is required to switchtowards a Th2-type response (30).

Despite the induction of high serum IgG titers to OVA in mice immunizedwith CT or ML-I+OVA, serum IgA was barely detectable. Previous workfound that immunogen-specific serum IgA was not detected in mice aftertwo intranasal immunizations with V. cholerae zot protein or LT+OVA(19). Similarly, it was found that oral delivery of LT+TT stimulatedhigh levels of serum IgG antibodies to TT, while anti-TT serum IgA wasnot detected (23). While both CT and ML-I effectively stimulatedanti-OVA IgA in all mucosal secretions, the levels were highest innasotracheal washes and saliva. Because the total serum IgA titers inmice immunized with CT+OVA and ML-I+OVA were 33-fold and 73-fold higherthan in nasotracheal washes and 1340-fold and 1176-fold higher than insaliva, respectively, the OVA-specific IgA titers at these sitesindicate the induction of local responses. Antibody titers in vaginalwashes were highly variable, which may reflect hormonal influences (31).TABLE 1 Serum IgG and IgG subclass titer IgA titer Antigen IgG 1gG11gG2a 1gG2b 1gG3 Serum saliva vagina nasal gut CT 532481 339841 832013681 1201 4481 409.6 358.8 3200 211.2 ML-I 337961 409601 2881 901 — 184164 440.3 819.2 33.6 LEA 61441 78081 361 11 — 361 11 10.2 49.6 5.2 PHA8561 7161 — — — 211 — — 4.6 0.2 WGA 141 21 — — — 51 0.2 0.6 1.4 — UEA-I10881 18441 11 — — 31 1.8 1 5 —

EXAMPLE 7 Three Different Lectins from the European Mistletoe (Viscumalbum), ML-I, ML-II, and ML-III, Increase the Titers of gD2-SpecificSerum IgG Antibodies after Intranasal Administration

One microgram of each of these three lectins was admixed with 5:gglycoprotein D2 (gD2) from Herpes simplex virus type 2 and deliveredintranasally to mice on days 1, 14, 28, and 49, as described above.Other mice were immunized intranasally with 5:g gD2 alone or with 5:ggD2 admixed with 1:g CT. Sera were collected 1 day before eachimmunization and at the termination of the study. Titers of gD2-specificserum IgG antibodies were measured as described above.

The results are shown in FIGS. 6A-D. Points refer to individual data,and the symbol (−) represents the mean titer. Each of the threemistletoe lectins exhibited adjuvant activity comparable to thatexhibited by CT.

EXAMPLE 8 Increases in the Titers of gD2-Specific Serum IgG SubclassAntibodies After Intranasal Administration

Mice were immunized intranasally on days 1, 14, 35, and 49 with eithergD2 alone (5:g) or gD2 (5:g) together with CT (1:g), ML-I (1:g), ML-II(1:g), or ML-III (1:g). Samples were collected two weeks after the finalimmunization. Data are titers measured two weeks after the finalimmunization. FIG. 7A, IgG1; FIG. 7B, IgG2a; FIG. 7C, IgG2b; FIG. 7D,IgG3. Points refer to individual data and the symbol (−) represents themean titer. p values in parentheses refer to significance of datacompared with the gD2 only group.

Titers of serum-specific IgG1, IgG2a, and IgG2b antibodies wereincreased in the mice treated with each of the three mistletoe lectins.

EXAMPLE 9 ML-I, ML-II, and ML-III Increase gD2-Specific IgA AntibodiesTiters in Mice After Intranasal Immunization

gD2-specific IgA antibody titers were measured in secretions of miceimmunized intranasally on days 1, 14, 35 and 49 with gD2 (5:g) alone orgD2 (5:g) together with CT (1:g), ML-I (1:g), ML-II (1:g) or ML-III(1:g). Data are titers measured two weeks after the final immunizationin FIG. 8. FIG. 8A, saliva; FIG. 8B, vaginal wash; FIG. 8C, nasotrachealwash; FIG. 8D, intestinal wash. Points refer to individual data and thesymbol (−) represents the mean titer. p values in parentheses refer tosignificance of data compared with the gD2 only group.

Each of the mistletoe lectins increased the titers of gD2-specific IgAantibodies in each of the secretions tested.

Table 2 shows lectin-specific antibody responses in mice immunizedintransally with gD2 (5:g), alone or together with CT/plant lectins(1:g). Mice (n=10) were immnnunized on days 0, 14, 28, and 42, andsamples were collected on days 56 and 57. TABLE 2 Serum IgG and IgGsubclass titer IgA titer Lectin/toxin IgG IgG1 IgG2a IgG2b IgG3 SerumSaliva Vagina nasal gut ML-I 313600 742400 2200 18400 — 2700 53 84 432 6ML II 655360 655360 3040 23040 — 520 98 34 166 30 ML III 179200 3276801220 9920 — 520 58 88 113 7 CT 523378 568889 176356 193422 211 7111 484409 1771 53

EXAMPLE 10 Mucosal Immunogenicity and Adjuvanticity of Nontoxic Type IIRIPs and Related Molecules

The mucosal (intranasal) immunogenicity and adjuvanticity of nontoxictype II RIP (Nigrin B, Basic Nigrin B, Ebulin r1) and molecules relatedto their B subunits (SNA II, SELfd) was compared with that of ML-1 andCT. Mice were immunized intranasally with gD2 (5:g) alone or togetherwith plant lectins or cholera toxin (CT) (1:g). Mice were immunized ondays 0, 21, and 42 and samples were collected on days 56 and 57.Lectin-specific responses and responses to the bystander antigen, gD2,were measured by ELISA.

FIG. 9 shows the gD2-specific total serum IgG and IgG subclass titersfrom mice immunized intranasally on days 1, 21, and 42 with either gD2(5:g) alone or gD2 (5:g) together with 1:g of CT, ML-I, Nigrin B, BasicNigrin B, Ebulin r1, SNA II or SELfd. Sera were collected at thetermination of the study. Points refer to individual data and the symbol(−) represents the mean titer. p values in parentheses refer tosignificance of data compared with the gD2 only group.

FIG. 10 shows gD2-specific IgA antibody titers measured in secretions ofmice immunized intranasally on days 1, 21, and 42 with either gD2 (5:g)alone or gD2 (5:g) together with 1:g of CT, ML-I, Nigrin B, Basic NigrinB, Ebulin r1, SNA II or SELfd. Data are titers measured two weeks afterthe final immunization in (a) saliva, (b) vaginal wash, (c) nasotrachealwash, (d) intestinal wash. Points refer to individual data and thesymbol (−) represents the mean titer. p values in parentheses refer tosignificance of data compared with the gD2 only group.

Table 3 shows the immunogenicity of type II RIP and related molecules.Antibody responses were measured in mice immunized intranasally with gD2(5:g) alone or together with CT/lectins (1:g). Groups of mice (n=10)were immunized on days 0, 14, 28 and 42 and samples were collected ondays 56 and 57. TABLE 3 Lectin/ Serum IgG and IgG subclass titer IgAtiter toxin IgG IgG1 IgG2a IgG2b IgG3 Serum Saliva Vagina Nasal Gut ML-I921600 1556480 6820 96000 10 1880 28.8 421 716.8 10 Nigrin B 120 880 2060 — 10 0.2 0.4 — 0.6 Basic — 10 — — — — — — — — Nigrin B Ebulin r1 59209100 480 6160 — 170 1.6 0.2 4.4 — SNA II 40 50 — 40 — 80 — — 1.4 1 SELfd310 750 — 90 — 150 — — — —

FIG. 11 shows gD2-specific serum IgA and IgG antibody titers measured inmice immunized intranasally on days 1, 21, and 42 with either gD2 (5 μg)alone or gD2 (5 μg) together with 1 μg of CT, ML-I, or UEA-1. Data aretiters measured two weeks after the final immunization. Points refer toindividual data and the symbol (−) represents the mean titer. p valuesin parentheses refer to significance of data compared with the gD2 onlygroup.

FIG. 12 shows gD2-specific IgG subclass antibody titers measured in miceimmunized intranasally on days 1, 21, and 42 with either gD2 (5 μg)alone or gD2 (5 μg) together with 1 ∞g of CT, ML-I, or UEA-1. Data aretiters measured in sera two weeks after the final immunization. Pointsrefer to individual data and the symbol (−) represents the mean titer. pvalues in parentheses refer to significance of data compared with thegD2 only group.

FIG. 13 shows gD2-specific IgA antibody titers measured in secretions ofmice immunized intranasally on days 1, 21 and 42 with either gD2 (5 μg)alone or gD2 (5 μg) together with 1 μg of CT, ML-I, or UEA-1. Data aretiters measured two weeks after the final immunization in (a) saliva,(b) vaginal wash, (c) nasotracheal wash, (d) intestinal wash. Pointsrefer to individual data and the symbol (−) represents the mean titer. pvalues in parentheses refer to significance of data compared with thegD2 only group.

FIG. 14 shows mean concentrations of IL-5, IL-4, and IFN production andcounts per minute for T-cell proliferation assay in (a) spleen cells and(b) cervical lymph nodes at week 8 after three immunizations (days 0,21, 42) with gD2, ML-1 or UEA-1 or with gD2 with ML-1, UEA-1 or LTK63.Spleen cells and cervical lymph node cells were isolated and stimulatedin vitro with gD2 (0 μg/ml, 1 μg/ml, or 5 μg/ml) or with gD2 coupled tolatex beads diluted 1:1000 or 1:5000 or with PMA/cd3.

FIG. 15 shows OVA-specific serum IgG antibody titers from mice immunizedby gavage on days 1, 14, 28 and 49 with either OVA (5 mg) alone or OVA(5 mg) together with CT (10 μg), ML-I (10 μg), ML-II (10 μg) or ML-III(10 μg). Sera were collected 1 day before each immunization and at thetermination of the study. FIG. 1 5A, serum IgG titers after one dose(day 13); FIG. 15B, serum IgG titers after two doses (day 27); FIG. 15C,serum IgG titers after three doses (day 48); FIG. 15D, serum IgG titersafter the final dose (day 62). Points refer to individual data, and thesymbol (−) represents the mean titer.

FIG. 16 shows OVA-specific serum IgG subclass and IgA antibody titersmeasured in mice immunized by gavage on days 1, 14, 35, and 49 witheither OVA (5 mg) alone or OVA (5 mg) together with CT (10 μg), ML-I (10μg), ML-II (10 μg), or ML-III (10 μg). Samples were collected two weeksafter the final immunization. Data are titers measured two weeks afterthe final immunization. FIG. 16A, IgG1; FIG. 16B, IgG2a; FIG. 16C,IgG2b; FIG. 16D, IgG3. Points refer to individual data, and the symbol(−) represents the mean titer.

FIG. 17 shows OVA-specific IgA antibody titers measured in secretions ofmice immunized by gavage on days 1, 14, 35 and 49 with OVA (5 mg) aloneor OVA (5 mg) together with CT (10 μg), ML-I (10 μg), ML-II (10 μg), orML-III (10 μg). Data are titers measured two weeks after the finalimmunization. FIG. 17A, saliva; FIG. 17B, vaginal wash; FIG. 17C,nasotracheal wash; FIG. 17D, intestinal wash. Points refer to individualdata, and the symbol (−) represents the mean titer.

Table 4 shows OVA-specific antibody responses in mice immunized bygavage with OVA (5 mg) alone or together with lectins (10 μg)administered in 0.5 ml sodium bicarbonate. Groups of mice (n=10) wereimmunized on days 0, 14, 28, and 42, and samples were collected on days56 and 57. TABLE 4 Serum IgG and IgG subclass titer IgA titerLectin/toxin IgG IgG1 IgG2 IgG2b IgG Serum Saliva Vagina Nasal gut OVAonly 30500 62260 — 120 — 40 — — 0.4 — ML I + OVA 361333 625867 168916267 — — 8 6 43 4 ML II + OVA 290800 462400 533 4367 — 133 1 3 9 8 MLIII + OVA 362000 603200 1300 15700 — 200 15 8 100 17 CT + OVA 401067534756 2311 7578 — 1375 11 10 119 26

Table 5 shows OVA-specific antibody responses in mice immunized with OVA(5 mg) alone or together with lectins (10 μg). Mice were administeredwith the antigen (±ML1) either by gavage in 0.1 ml PBS or incorporatedin the feed pellet. Groups of mice (n=5) were immunized on days 0, 14,28, and 42, and samples were collected on days 56 and 57.

Table 6 shows ML1-specific antibody responses in mice immunized orallywith OVA (5 mg) alone or together with lectins (10 μg). Mice wereadministered with the antigen (±ML1) either by gavage in 0.1 ml PBS orincorporated in the feed pellet. Mice were immunized on days 0, 14, 28,and 42, and samples were collected on days 56 and 57. TABLE 5 Serum IgGand IgG subclass titer IgA titer Lectin IgG IgG1 IgG2a IgG2b IgG3 SerumSaliva Vagina nasal gut OVA only 6600 (4) 25640 (3) 160 (1) 3240 (3) — —— 3.2 (1) — — ML I + OVA (PBS) (PBS) 6120 (5)  8340 (5) — — — 80 (1) — ——   2 (2) OVA ONLY (PELLET)  20 (1)   40 (1) — — — — — — — — ML I + OVA(PELLET)  160 (3)   20 (1) —  40 (2) — — — — — 0.4 (1)

TABLE 6 Serum IgG and IgG subclass titer IgA titer Lectin IgG IgG1 IgG2aIgG2b IgG3 Serum Saliva Vagina Nasal Gut OVA ONLY (PBS) ML I + OVA (PBS)6080 (5) 33280 (5) 240 (2) 4640 (5) — 1440 (5) 0.4 (1)   2 (3) 0.4 (1)  16 (5) OVA ONLY PELLET) ML I + OVA (PELLET) 6600 (5) 31040 (5) 120 (2)2260 (5) —  560 (4) — 35.2 (4) 0.8 (1) 3..2 (3)

EXAMPLE 11 Efficacy of Type II RIP (ML1, Ebulin R1) as Adjuvants WhenDelivered with Antigens by the Transcutaneous Route

Following on from studies that demonstrated the efficacy of mistletoelectins as mucosal adjuvants, these studies were carried out to assessthe potential of type II RIP as adjuvants when administeredtranscutaneously. Recent work has demonstrated the effective inductionof immune responses when CT is used as an adjuvant by this route (Glennet al., 1998, 1999). In addition to ML 1, CT was used as a positivecontrol and Ebulin r1 because it was the most immunogenic of thenontoxic type II RIP when administered intranasally.

Protocol

Groups of female Balb/c mice (n=5) were immunized on days 0 and 21 andserum samples were taken on days 0, 20, and 35 for analysis by ELISA.Three different bystander antigens, BSA, DT and gD2 were investigated.Antigens (50 μg) were administered to mice either alone or mixed withlectin/toxin (50 μg). Specific antibody responses were determined byELISA. Additionally, the responses to CT and lectins was measured toassess their immunogenicity by the transcutaneous route. The backs ofmice were shaved with a no. 40 clipper and animals were allowed to restfor 48 hr. Mice were anesthetized with hypnorm-diazepam during theimmunization procedure. The skin was swabbed with ethanol 1 min prior toapplication of solution. Immunizing solution (100 μl) was applied toshaved skin over a 2 cm² area. After 30 min, a further 100 μl ofdistilled water was applied and mice were left for 90 min. Mice wereextensively washed with lukewarm tap water, patted dry, and washedagain.

Study Groups

I. BSA

-   1. BSA 50:g-   2. BSA 50:g+CT 50 μg-   3. BSA 50:g+ML I 50 μg-   4. BSA 50:g+Ebulin r1 50:g    II. Diphtheria Toxoid (DT)-   1. DT 50:g-   2. DT 50:g+CT50 μg-   3. DT 50:g+MLI 50 μg-   4. DT 50:g+Ebulin rI 50:g    III. Herpes Simplex Virus 2 Glycoprotein D (gD2-   1. gD2 50:g-   2. gD2 50:g+CT 50 μg-   3. gD2 50:g+ML I 50 μg-   4. gD2 50:g+lectin II 50:g

Table 7 shows BSA-specific serum antibody titers measured following 1and 2 transcutaneous doses of BSA (50:g) alone or together with CT/plantlectin (50:g). TABLE 7 Serum Serum Serum IgG IgG IgG2a Adjuvant/ Mousetiter titer Serum IgG1 titer antigen number week 3 week 5 titer week 5week 5 BSA 1 100 100 100 <100 2 <100 6400 6400 <100 3 <100 25600 25600<100 4 <100 1600 1600 <100 5 <100 12800 12800 <100 CT + BSA 1 <100819200 819200 <100 2 3200 1638400 1638400 12800 3 6400 409600 4096001600 4 1600 819200 819200 3200 5 400 819200 819200 800 ML1 + BSA 1 800102400 102400 <100 2 1600 204800 204800 1600 3 1600 204800 204800 400 4<100 409600 409600 <100 5 <100 204800 204800 200 Ebulin r1 + BSA 1 <100102400 102400 <100 2 800 204800 25600 <100 3 200 204800 102400 <100 4<100 409600 204800 <100 5 <100 204800 204800 <100

Table 8 shows DT-specific serum antibody titers measured following 1 and2 transcutaneous doses of DT (50:g) alone or together with CT/plantlectin (50:g). TABLE 8 Serum Serum Serum IgG IgG IgG1 Adjuvant/ Mousetiter titer titer Serum IgG antigen number week 3 week 5 week 5 titerweek 5 DT 1 100 <100 200 <100 2 <100 25600 102400 <100 3 <100 1280025600 100 4 100 800 3200 <100 5 <100 100 400 <100 CT + DT 1 256001638400 1638400 800 2 12800 1638400 1638400 1600 3 25600 3276800 65536003200 4 25600 1638400 3276800 3200 5 51200 819200 1638400 1600 ML1 + DT 1800 51200 102400 <100 2 100 25600 102400 100 3 <100 6400 12800 <100 4<100 51200 102400 100 5 <100 6400 12800 100 Ebulin r1 + DT 1 <100 400800 <100 2 800 100 200 <100 3 1600 102400 204800 100 4 400 12800 25600<100 5 <100 51200 204800 <100

Table 9 shows gD2-specific serum antibody titers measured following 1and 2 transcutaneous doses of gD2 (50:g) alone or together with CT/plantlectin (50:g). Due to the gGa and IgG1 levels were not determined. TABLE9 Adjuvant/ Mouse Serum IgG Serum IgG antigen number titer week 3 titerweek 5 gD2 1 <100 <100 2 100 100 3 <100 <100 4 <100 <100 5 <100 <100CT + gD2 1 100 1600 2 <100 102400 3 <100 3200 4 <100 3200 5 <100 6400ML1 + gD2 1 <100 <100 2 <100 <100 3 <100 1600 4 <100 <100 5 <100 <100Ebulin r1 + gD2 1 <100 <100 2 <100 <100 3 100 <100 4 100 200 5 100 <100

Table 10 shows CT and lectin-specific serum antibody titers measuredfollowing 1 and 2 trancutaneous doses with CT/ML1/Ebulin r1 (50:g).TABLE 10 Mouse Serum IgG Serum IgG Lectin/toxin number titer week 3titer week 5 CT 1 3200 102400 2 3200 51200 3 3200 12800 4 <100 204800 5204800 1638400 6 12800 102400 7 12800 204800 8 3200 204800 9 3200 10240010 800 204800 11 <100 102400 12 <100 819200 13 <100 102400 14 800 40960015 800 102400 ML 1 1 800 1600 2 400 3200 3 400 1600 4 800 51200 5 <10025600 6 <100 <100 7 <100 400 8 <100 <100 9 <100 800 10 <100 <100 11 <10012800 12 <100 1600 13 <100 1600 14 <100 25600 15 <100 200 Ebulin r1 1<100 400 2 100 <100 3 100 <100 4 800 200 5 200 <100 6 100 <100 7 100<100 8 <100 100 9 <100 <100 10 <100 <100 11 <100 <100 12 <100 <100 13<100 100 14 <100 400 15 100 <100

EXAMPLE 12 Binding, Uptake and Translocation of Orally Delivered Lectinsin Mice

Administration of lectins and collection of tissues. Female Balb/c micewere maintained on a normal stock diet with free access to water priorto experiments. Mice were deprived of food overnight and lectins weredelivered by gavage using curved oral dosing needles (20 g×25 mm) (1 mglectin/mouse in 100 μl physiological saline) to groups of 24 mice. Waterwas available throughout. Groups of 8 animals were sacrificed byhalothane anesthesia followed by exsanguination after 1 hr, 6 hr and 24hr. Blood was collected by cardiac puncture. Mice were dissected, andthe entire gut was removed and divided into stomach, two parts of smallintestine and large intestine. Gut contents were washed out with 10 mlof ice-cold PBS to give an indication of the amount of unbound lectinpresent. Gut tissues were placed in polythene bags and snap frozen inliquid nitrogen. Sections of intestine were taken in each case and fixedin 4% formalin for examination of lectin binding by histology. All guttissues and washings were stored at −20° C. until required for analysis.Additionally, the liver, spleen and kidneys were collected from animals.

Extraction of Lectins from Tissue.

Tissues from animals administered with lectins or with control salinewere extracted by homogenization in a 20 mM solution of diaminopropane.Tissue pieces were placed in the extracting solution (995 μl 20 mMdiaminopropane+5 μl of 5 mg ml⁻ Aprotinin (Sigma)) and homogenized(Janke and Kunkel IKA®-Labortechnik, Ultra-Turrax®) at 24000 rpm for 2minutes on ice. The homogeniser head was washed with distilled water, in1 ml of extracting solution and again in distilled water betweensamples. Samples was centrifuged (Jouan, MRI 22) for 20 min at 18600 gat 2° C. The supernatants were collected and stored at −20° C. untilrequired for analysis.

Processing of Gut Washing.

To provide an indication of the amount of free (unbound) lectin presentin the gut, the amount of lectin present in gut washings was analysed.Washings (500 μl) were added to dilution buffer (495 μl)+the proteaseinhibitor Aprotinin (5 μl of 5 mg ml⁻¹) and centrifuged (Microspin 12S,Sorvall® Instruments, Du Pont) at 8000 rpm for 10 min.

Processing of Blood Samples.

After collection, blood samples were left at room temperature for 1 hourand centrifuged at 7000 rpm for 6 min (Microspin 12S, Sorvall®Instruments, Du Pont). Plasma was collected and stored at −20° C. untilrequired for analysis.

ELISA Analysis of Binding of Plant Lectins to the Gut.

An ELISA assay was set up to enable the quantification of WGA inextracted tissue samples and washings. Microtiter plates (Immunolon 4,Dynatech) were coated with 75 μl a 1:64000 dilution of rabbit anti-WGAper well in carbonate-bicarbonate buffer, pH 9.6 and incubated at 4° C.overnight. After washing, plates were blocked with PBST/2% gelatin/200mM N-acetylglucosamine and incubated at 37° C. for 1 hr. Plates werewashed; standards and samples added, serially diluted in dilution buffer(PBST/200 mM N-acetylglucosamine) and incubated at 37° C. for 1 hr. Astandard curve for WGA was constructed by titrating a WGA solution from10 ng/ml to 78 pg/ml. Biotinylated anti-WGA at a dilution of 1:16000 indilution buffer was added and incubated at 37° C. for 1 hr. Afterfurther washes, ExtrAvidin® peroxidase (Sigma) at a dilution of 1:1000,in dilution buffer was added and incubated at 37° C. for 30 min. Plateswere washed and 50 μl/well of developing solution (TMB microwellperoxidase substrate (1-C) Kirkegaard and Perry Laboratories,Gaithersburg, USA) was added and incubated in the dark with shaking at37° C. for 30 min. The reaction was stopped by addition of 1 M H2SO4 (50μl/well) and the absorbance read at 450 nm. WGA levels were calculatedfrom the linear region of the standard curve.

SDS-PAGE and Western Blotting. SDS-PAGE gels were run and proteinstransferred to PVDF membranes using a semi-dry transfer apparatus. Aftertransfer, membranes were blocked in a 2.5% casein solution for 30 min atroom temperature. Membranes were washed and the primary antibody(biotin-labeled anti-lectin) was added at a dilution of 1:2500 in 5 mlof 1.2% casein solution. Following incubation with agitation at roomtemperature overnight, membranes were washed extensively with PBS andExtrAvidin® peroxidase added at a 1:5000 dilution in 1.2% casein.Following a 1 hr incubation at room temperature, membranes were washedextensively with PBS and distilled water. Excess fluid was blotted frommembranes and the developing solution was added (Super signal® West picodetection kit (Pierce, Rockford, USA)) and left in the dark for 5 min.Excess fluid was dried from membranes and membranes were exposed to film(Kodak X-OMAT LS (Sigma)) and processed.

Results

Stability and Binding of Plant Lectins in the Mouse Gut Following OralGavage.

The lectins, PHA, WGA and UEA-1 were stable in the mouse digestive tractfor up to 6 hr after gavage (Tables 11 and 12; FIGS. 18 and 19). In thesmall intestine, the lectins were only detected at the subunit MW of thepositive control. In the stomach there was an indication of lectinaggregation after 6 and 24 hr in the cases of PHA and UEA-1. However,most of the lectin detected in the stomach was also intact. Analysis oflectin binding to the gut found differences in the location of lectinbinding at 1 and 6hr after delivery. PHA (and WGA, not presented) boundto the proximal small intestine while UEA-1 was not detected in thisregion but bound to the distal small intestine (Table 11). The patternof binding was similar at 1 and 6 hr after lectin administration. At 24hr after delivery, lectins were not detected in homogenised gut tissues.This indicated that lectins did not detach from the gut and re-bind butmore likely were excreted after detachment.

Table 11 shows detection of PHA (isotype E2L2) in the mouse digestivetract at 1, 6, and 24 hr after the delivery of 1 mg by gavage. The +symbol indicates that lectin was detected on Western blots and thenumber of mice with a positive signal (out of 8 in each case) ispresented in parentheses. The positive control PHA subunit molecularweight was 29.5 kDa.

Table 12 shows detection of UEA1 in the mouse digestive tract at 1, 6,and 24 hr after the delivery of 1 mg by gavage. The + symbol indicatesthat lectin was detected on Western blots and the number of mice with apositive signal (out of 8 in each case) is presented in parentheses. Thepositive control UEA1 apparent subunit molecular weight was 34.7 kDa.TABLE 11 TIME AFTER DELIVERY MW(kDa) GUT REGION (hr) 125.9 114.8 91.258.1 43.7 29.5 9.3 7.6 Gutwash 1 − − − − − + (8) − − 6 − − − − − − − −24 − − − − − − − − Stomach 1 − − − + (7) − + (8) − − 6 − − − + (3) − − −− 24 − − − − − − + (4) + (4) Small intestine 1 − − − − − + (8) − −Homogenate 6 − − − − − + (8) − − (Proximal) 24 − − − − − − − − Smallintestine 1 Homogenate 6 − − − − − + (6) − − (Distal) 24 − − − − − − − −Large intestine 1 − − − − − − − Homogenate 6 − − − − − − − 24 − − − − −− −

TABLE 12 TIME AFTER GUT DELIVERY APPARENT MOLECULAR WEIGHT (kDa) REGION(hr) 144.5 114.8 100 87.1 75.9 64.6 52.5 34.7 Stomach 1 − − − − − − − +(4) 6 − − − + (4) + (3) + (3) − − 24 − − − + (1) − + (3) − − Smallintestine 1 − − + (2) − + (1) − − + (4) Wash 6 − − − − − − − − 24 − − −− − − − − Small intestine 1 − − − − − − − − homogenate 6 − − − − − − − −(Proximal) 24 − − − − − − − − Small intestine 1 − − − − − − − + (4)homogenate 6 − − − − − − − + (5) (Distal) 24 − − − − − − − − Largeintestine 1 − − − − − − − + (2) homogenate 6 − − − − − − − + (4) 24 − −− − − − − −

Detection of Lectins in Internal Organs and Blood.

Sensitive chemiluminescent Western blotting assays were used todetermine lectin uptake. For all three lectins, uptake into the liverand kidney was measured (Table 13, FIGS. 20 and 21). FIG. 20 shows thatUEA1 detected in kidney tissue are at a higher molecular weight than inthe positive control. There is a cross reaction with control kidneytissue. However, additional bands are visible in mouse tissues fromanimals administered with lectin. These bands are at a higher molecularweight than in the control.

Lectins were detected in liver tissue from 1 to 24 hr afteradministration. However, the MW of the reactive bands was considerablyhigher than the expected subunit MW. In fact, none of the 3 lectins weredetected at the expected subunit WM in internal organs. To get anindication of the degree of lectin uptake, a sandwich ELISA was set upto quantify WGA. This enabled a determination of lectins in the gut andinternal organs (Table 13). The lectin was detected in gut homogenatesfor up to 6 hr after delivery at approximately the level of lectinadministered. At 24 hr, no lectin was detected in homogenates. Of theinternal organs, the highest levels of lectin were detected in livertissue. The level of lectin at this site increased from 1 to 24 hr.However, the highest amount of lectin detected (289.3 ng) represented asmall fraction of the delivered dose. Very low levels of WGA weredetected in the blood cells or sera or in the spleen. The detection ofthe highest level of lectin in the liver and kidney is in line with theWestern blotting results where the lectins were detectable in the liverand kidney tissue but not in blood or the other organs.

Stability of Native and Absorbed Lectins to Proteolysis by Trypsin.

To determine if the absorbed lectins detected in the liver and kidneysretained the properties of the native lectins, native andtissue-extracted PHA, WGA and UEA-1 were incubated for 1 hr with asolution of trypsin. All three lectins were highly stable to the enzymein their native form. However, the modified lectins detected in liverand kidney tissue were degraded by the enzyme (FIG. 22). This indicatesthat cells in the liver and kidney are capable of modifying plantlectins to forms which are sensitive to proteolysis. This may be amechanism for degradation of ingested plant lectins which survive in thedigestive tract and are absorbed.

TABLE 13 shows detection of WGA in tissues following oral gavage ofgroups of mice (n=8) with a single dose of 1 mg lectin in 0.1 ml PBS.The lectin was determined by a quantitative sandwich ELISA and the dataare presented as gg lectin per organ or per ml for blood. TABLE 13 TIMEAFTER LECTIN DELIVERY (hr) TISSUE 1 6 24 Gut homogenate ND 1018.1 ±949.2  — Gut wash 5.5 ± 5.8 2.5 ± 1.4 0.4 ± 0.7 Liver 28.5 ± 33     48 ±42.1 289.3 ± 546   Spleen 0.6 ± 0.9 3.3 ± 2.1 11.9 ± 9.2  Kidney 0.5 ±0.5 1.9 ± 0.8 1.6 ± 2.2 Serum 0.4 ± 0.7 2.1 ± 1.8 1.1 ± 2   Blood cells0.3 ± 0.5 1.8 ± 2.5 0.5 ± 0.8References

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1. A method of producing an immune response in a mammal, comprising the step of: administering to a mammal an admixture comprising an immunogen and a plant lectin, whereby the mammal produces an immune response to the immunogen which is greater relative to the immune response to the immunogen produced in the absence of the plant lectin.
 2. The method of claim 1 wherein the admixture is administered mucosally.
 3. The method of claim 2 wherein the admixture is administered intranasally.
 4. The method of claim 1 wherein the plant lectin is selected from the group consisting of ML-1, ML-II, ML-III, WGA, and UEA-1.
 5. The method of claim 1 wherein the mammal is selected from the group consisting of a dog, a cat, a mouse, a rat, a rabbit, a guinea pig, a chimpanzee, a baboon, and a human.
 6. The method of claim 1 wherein the immune response is a T cell response.
 7. The method of claim 6 wherein the T cell response is a Th2 response.
 8. The method of claim 6 wherein the T cell response is proliferation of T cells.
 9. The method of claim 1 wherein the immune response is an antibody response.
 10. The method of claim 9 wherein the mammal produces an antibody which is selected from the group consisting of IgG and IgA antibodies.
 11. The method of claim 10 wherein the IgG antibodies are selected from the group consisting of IgG1, IgG2a, and IgG2b.
 12. The method of claim 10 wherein the antibodies are detectable in serum.
 13. The method of claim 10 wherein the antibodies are detectable in a mucosal secretion.
 14. The method of claim 13 wherein the mucosal secretion is obtained from a mucosa selected from the group consisting of gut mucosa, vaginal mucosa, oral mucosa, and nasal mucosa.
 15. The method of claim 1 wherein the admixture comprises two or more lectins.
 16. The method of claim 1 wherein the admixture comprises two or more immunogens.
 17. The method of claim 1 wherein the immunogen is a protein of an infectious agent.
 18. The method of claim 20 wherein the infectious agent is a virus.
 19. The method of claim 21 wherein the immunogen is a glycoprotein D2 protein from a Herpes simplex virus type
 2. 20. The method of claim 3 wherein the admixture is administered using a nasal spray.
 21. The method of claim 3 wherein a drop of a liquid containing the admixture is administered.
 22. The method of claim 1 wherein at least two doses of the admixture are administered.
 23. The method of claim 1 wherein the admixture comprises an immunogen and a plant lectin in a ratio of at least about 1:1.
 24. The method of claim 26 wherein the ratio is at least about 10:1.
 25. The method of claim 9 wherein an antibody titer is measured using an ELISA.
 26. The method of claim 1 wherein the admixture is administered by a method selected from the group consisting of oral administration, intranasal administration, intrarectal administration, vaginal administration, subcutaneous injection, intramuscular injection, transdermal injection, and transcutaneous injection.
 27. The method of claim 1 wherein the plant lectin is a type 2 ribosome inactivating protein.
 28. The method of claim 30 wherein the type 2 ribosome inactivating protein is selected from the group consisting of nigrin b, basic nigrin b, ebulin 1, ebulin r1, ebulin r, ebulin f, nigrin f, SNA1, SNA1′, SNAV, SNAVI, Sambucus nigra SNLRP1, SNLRP2, ricin, Ricinus lectin, Polygonatum RIP, Sieboldin-6, abrin, abrin 11, modeccin, volkensin, SSA, Cinnamonin, porrectin, gelorin, Evanthis hyemalis, RIP, Iris agglutinin, ML-I, ML-II, and ML-III.
 29. The method of claim 1 wherein the admixture is administered using a microparticle carrier.
 30. The method of claim 1 wherein the admixture is administered in conjunction with a bioadhesive polymer.
 31. The method of claim 1 wherein the admixture is in an enteric formulation. 